Lv v-haemorphin-7 neuroactive peptide and methods of use

ABSTRACT

The invention relates to neuroactive peptides or analogues thereof, having at least one of the biological activities of angiotensin IV, and which comprise the sequence Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, to methods of modulating neuronal activity, and to pharmaceutical composition thereof.

[0001] This invention relates to neuroactive peptides, and in particularto peptides which have the ability to act as analogues of angiotensinIV. The peptides of the invention bind with high affinity andspecificity to a variety of sites in the central nervous system, and areuseful as modulators of motor and cognitive function, and of neuronaldevelopment.

BACKGROUND OF THE INVENTION

[0002] The renin-angiotensin system has diverse roles in the regulationof body fluid and electrolyte balance and blood pressure control. Theseactions are exerted in a variety of target organs, including thecardiovascular system, adrenal glands, kidney and central and peripheralnervous systems, by both the circulating hormone and hormone locallyproduced in tissues. Most of these actions are exerted by theoctapeptide, angiotensin II, although the C-terminal heptapeptideangiotensin III has some activity. The hexapeptide NH2-Val Tyr Ile HisPro Phe-COOH, corresponding to the 3-8 fragment of angiotensin II (ie.amino acids 3-8), is also called angiotensin IV (Ang IV), and has untilrecently been believed to be an inactive degradation product devoid ofbiological activity.

[0003] However, Harding and co-workers have confirmed an earlier report(Braszko et al, 1988) that Ang IV has central nervous system activity,and can modify learning and behaviour (Wright et al, 1995). In addition,Ang IV has vasoactive effects, and can dilate cerebral arteries (Haberlet al, 1991) and increase renal blood flow (Swanson et al, 1992). This,coupled with the discovery of highly specific, high affinity sites forAng IV binding in bovine adrenal and other tissues, has reawakenedinterest in the hexapeptide, and the subject has been comprehensivelyreviewed (Wright et al 1995).

[0004] Ang IV has been associated with the central nervous systemeffects of increasing stereotypy behaviour (Braszko et al, 1988) andfacilitating memory retrieval in passive avoidance studies (Braszko etal, 1988; Wright et al, 1995). Ang IV also dilates cerebral arterioles(Haberl et al, 1991), and increases renal blood flow (Swanson et al,1992).

[0005] Receptor autoradiographic studies have revealed a widely abundantbut selective and characteristic distribution of binding sites for[¹²⁵I]Ang IV (known as the AT₄ receptor) in the guinea pig, sheep andmonkey central nervous systems, in regions associated with cholinergicneurons and in somatic motor and sensory associated areas (Miller-Winget al, 1993; Moeller et al, 1995, Moeller et al, 1996). In addition, AngIV binding sites are abundant in supraspinal components of the autonomicnervous system, and in the spinal cord are found in sympatheticpreganglionic neurons, in the dorsal root ganglia, and in Lamina II ofthe dorsal horn, and in the motor neurons of the ventral horn (Moelleret al, 1995).

[0006] The distribution of the Ang IV binding site differs from thelocalization of the Ang II AT₁ or AT₂ receptors. In addition, thepharmacology of each receptor is distinct in that the Ang IV siteexhibits a low to very low affinity for [Sar¹Ile⁸]Ang II, thenon-subtype selective Ang II antagonist, and losartan (du Pont-Merck)and PD 123319 (Parke-Davis), the specific AT₁ and AT₂ receptorantagonists respectively (Miller-Wing et al, 1993; Swanson et al, 1992;Hanesworth et al, 1993). Conversely, Ang II receptors show a lowaffinity for the Ang IV binding site (Bennett and Snyder, 1976).

[0007] The wide distribution of the Ang IV binding site in motor,sensory and cholinergic regions suggests important roles for thispeptide in the central nervous system. However, a physiological actionof the peptide in neurons has yet to be clearly defined.

[0008] Numerous neurotransmitters and neuropeptides have been associatedwith the regulation of neuronal development. Acetylcholine inhibitsneurite outgrowth from embryonic chicken ciliary ganglion cells andsympathetic neurons (Pugh and Berg, 1994; Small et al, 1995), and rathippocampal neurons (Muttson, 1988). Conversely, vasoactive intestinalpeptide stimulates superior cervical ganglion branching (Pincus et al,1990) and somatostatin increases neuronal sprouting from Helisoma buccalganglion neurons (Bulloch, 1987).

[0009] We have now surprisingly found that the peptide LVV-haemorphin-7,derived from β-globin, acts as an agonist at the AT₄ receptor, and isthe endogenous ligand for the AT₄ receptors in the brain. We havecharacterised its pharmacological activity. This enables us to designnovel agonists and antagonists of Ang IV action.

SUMMARY OF THE INVENTION

[0010] According to a first aspect, the invention provides a method ofmodulating motor neuron activity, cholinergic neuron activity, orneuronal development, comprising the step of administering an effectiveamount of a neuroactive peptide having at least one of the biologicalactivities of angiotensin IV as herein defined, comprising the aminoacid sequence: Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, (SEQ ID NO:1) ora biologically-active analogue or fragment of said peptide to a mammalin need of such treatment. This aspect of the invention specificallyincludes the use of decapeptide sequence referred to above in the methodof the invention which relies on a previously unknown and unsuspectedactivity of the decapeptide.

[0011] It will be clearly understood that the sequence of the inventionmay be modified by conservative amino acid substitutions, insertions,deletions or extensions, provided that the biological activity isretained. Such variants may, for example, include sequences comprisingD-amino acids, non-naturally occurring amino acids, and/or amino acidanalogues. Thus the analogue may be a peptidomimetic compound.

[0012] Preferably the mammal is a human.

[0013] The Ang IV agonist and antagonist compounds according to theinvention are useful in the treatment of a variety of conditions,including but not limited to:

[0014] Dementia, including Alzheimer's disease

[0015] Other neurodegenerative disorders involving cholinergic pathways,motor pathways, or sensory pathways, such as motor neurone disease

[0016] sensory and motor peripheral neuropathies

[0017] brain or spinal cord injury due to trauma, hypoxia or vasculardisease.

[0018] In a second aspect, the invention provides a non-peptide analogueof the peptide of the invention. This non-peptide analogue is to beunderstood to encompass modifications or substitutions of the peptidestructure which are designed to improve the bioavailability, metabolicstability, half-life in the body, or to modify the biological activity,of the compound of the invention. Such non-peptide analogues are knownin the art, for example compounds in which the peptide backbone isreplaced by a non-peptide chain, and are often referred to aspeptidomimetic compounds. Alternatively, in one or more of the peptidelinkages the order of the nitrogen and carbon atoms can be reversed toform a pseudo peptide bond. One or more of the amino acid side-chainsmay be replaced by an analogous structure of greater stability. Manyother such variations will occur to the person skilled in the art. Theonly requirement is that the overall 3-dimensional structure issufficiently preserved that ability to bind to the AT₄ receptor atsuitable affinity is retained. Using modern methods of peptide synthesisand combinatorial chemistry, it is possible to synthesize and test verylarge numbers of analogues within a short space of time, and suchsynthesis and screening is routinely carried out by pharmaceuticalcompanies.

[0019] Considerable information is available regarding the structuralfeatures of Ang IV peptides which are necessary for high affinity, andthese results may be used as guidelines for modification of the peptidesof the invention. See for example Wright et al, 1995.

[0020] The person skilled in the art will appreciate that by modifyingthe sequence or by constructing a non-peptide analogue the activity ofthe compound of the invention can be very considerably modified. Notonly can improvement in activity be obtained, it is also possible toobtain compounds which bind to the AT₄ receptor in such a way that AngIV activity is inhibited. Such inhibitory compounds can have the abilityto antagonize the activity of Ang IV. The person skilled in the art willreadily be able to synthesize modified peptides and peptide analoguesand to test whether they have activity as Ang IV agonists orantagonists, using methods well known in the art.

[0021] According to a third aspect, the invention provides a method ofscreening for putative agonists or antagonists of the effect ofLVV-haemorphin-7 on neuronal activity, comprising the step of testingthe ability of the compound to stimulate or inhibit the effect ofLVV-haemorphin-7 on a biological activity selected from the groupconsisting of modifying learning, modifying behaviour, vasoactiveeffects, dilation of cerebral arteries, increase in renal blood flow,increase in stereotypy behaviour, facilitating memory retrieval, neuritemodelling and alleviation of the effects of spinal cord injury.

[0022] Thus according to a fourth aspect, the invention also providescompounds which are able to act as agonists or antagonists of theneuroactive peptides of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The invention will be now described in detail by way of referenceonly to the following non-limiting examples, and to the figures, inwhich

[0024]FIG. 1 shows competition curves derived from prefrontal corticalsections incubated with [¹²⁵I]Ang IV in the presence of increasingconcentrations of the following unlabelled ligands: ▴ Ang IV, □ Ang II,▪ Ang III, Δ Ang II(1-7),  losartan and ∘ PD 123319. Values are themean of four sections, each from two animals. B/Bo×100 expressed as apercentage available receptors occupied;

[0025]FIG. 2 shows the results of competition binding studies showingthe inhibition of [¹²⁵I]Ang IV binding to E13 chicken chorioallantoicmembranes with varying concentrations of unlabelled compounds: ▴ Ang IV,

Nle¹-AIV, Δ CGP 42112, □ Ang II, ▾ Nle¹-Y-I-amide,

WSU-4042, ▪ [Sar¹Ile⁸]Ang II,  PD 123319 and ∘ losartan. Values areexpressed as a percentage of total binding, and are pooled from twoexperiments. B/Bo×100=% of available receptors occupied;

[0026]FIG. 3 summarizes competition binding studies showing theinhibition of ¹²⁵I[Sar¹Ile⁸]Ang II binding to E13 chickenchorioallantoic membranes with varying concentrations of unlabelledcompounds: ▪ [Sar¹Ile⁸]Ang II, □ Ang II, Δ CGP 42112,

Nle¹-AIV, ▴ Ang IV, ∘ losartan,  PD 123319,

WSU-4042 and ▾ Nle¹-Y-I-amide. Values are expressed as a percentage oftotal bnding, and are pooled from two experiments. B/Bo×100=% ofavailable receptors occupied;

[0027]FIG. 4 shows the effect of Ang IV on neurite outgrowth from E11chicken sympathetic neurons. Values are expressed as a percentage ofcontrol levels, and are depicted as the mean±standard error of the mean(SEM). The results are pooled from 3 experiments, each with at least 40neurite measurements. * indicates a significant difference from controlvalues using Bonferroni's test;

[0028]FIG. 5 shows the effect of 10 nM Ang IV on neurite outgrowth inthe presence of 1 μM Nle¹-Y-I-amide, WSU-4042, Nle¹-AIV, [Sar¹Ile⁸]AngII, losartan, PD 123319 and CGP 42112. Values are expressed as apercentage of control levels, and are depicted as the mean±S.E.M. Theresults are pooled from 3 experiments, each with at least 40 neuritemeasurements. * indicates a significant difference from control valuesusing Bonferroni's test;

[0029]FIG. 6 shows the effect of 10 nM Ang II on neurite outgrowth inthe presence of 1 μM Nle¹-Y-I-amide, WSU-4042, Nle¹-AIV, [Sar¹Ile⁸]AngII, losartan, PD 123319 and CGP 42112. Values are expressed as apercentage of control levels, and are depicted as the mean+S.E.M. Theresults are pooled from 3 experiments, each with at least 40 neuritemeasurements. * indicates a significant difference from control valuesusing Bonferroni's test;

[0030]FIG. 7 illustrates the binding of ¹²⁵I-angiotensin IV to sheepspinal cord. The arrow indicates the site of damage to the spinal cord;

[0031]FIG. 8 summarizes the results of competition binding studiesshowing the inhibition of [¹²⁵I]LVV-haemorphin-7 binding to sheepcerebellar cortical membranes with varying concentrations of unlabelledcompounds: ▴ Ang IV, Δ LVV-haemorphin-7, ▪ Ang III, □ Ang II, ∘ PD123319,  losartan, * naloxone and ∇ haloperidol. Values are the mean ofthree experiments. B/Bo×100=% of available receptors occupied;

[0032]FIG. 9 summarizes the results of competition binding studiesshowing the inhibition of [¹²⁵I]Ang IV binding to sheep cerebellarcortical membranes with varying concentrations of unlabelled compounds:▴ Ang IV, Δ LVV-haemorphin-7, ▪ AngIII, □ AngII, ∘ PD 123319, losartan, * naloxone and ∇ haloperidol. Values are the mean of threeexperiments. B/Bo×100=% of available receptors occupied;

[0033]FIG. 10 is a schematic diagram illustrating the position of theoligonucleotide probes used for cloning and PCR experiments. (A)schematic diagram of the β-globin precursor showing relevant positionand direction of oligonucleotides used. The shaded region represents theLVV-haemorphin-7 sequence, which is given below. (B) sequences of theoligonucleotides H170 to H173 (SEQ ID Nos:2 to 5 respectively) used inthis study;

[0034]FIG. 11 illustrates the detection of β-globin mRNA by RT-PCR andSouthern blotting in sheep cerebellar and cerebral cortices, heart andliver. Molecular weight markers are shown on the left;

[0035]FIG. 12 shows the complete nucleotide sequence of Clone EX (SEQ IDNO:6); and

[0036]FIG. 13 shows the nucleotide sequence (SEQ ID NO:7) and derivedamino acid sequence of the rat EX clone. The region of the potentialLVV-haemorphin-7 is shown in bold.

[0037]FIG. 14 summarizes the effects of LVV-haemorphin-7 on theperformance of scopolamine-treated rats in a passive avoidance task.

[0038]FIG. 15 summarizes the effects of LVV-haemorphin-7 on theperformance of scopolamine-treated rats in a water maze acquisitiontrial.

[0039] The unlabelled ligands, Ang IV (Peninsula Laboratories, Calif.USA), Ang II and the Ang II antagonist [Sar¹Ile⁸]Ang II (Sigma, Mo.USA), the Ang II partial agonist CGP 42112 (Ciba-Geigy, BasleSwitzerland), the Ang II ATI antagonist, losartan (Du Pont MerckPharmaceutical Company, Del. USA), the Ang II AT₂ antagonist, PD 123319(Parke-Davis, Mich. USA-Ms. C. L. Germain), and the Ang IV analogues,WSU 4042, Nle¹-Y-I-amide and Nle¹-AIV (prepared as previously describedby Sardinia et al, 1993), were used at final concentrations ranging from10⁻⁹ to 10⁻⁴ M.

EXAMPLE 1 Mapping of Angiotensin AT₄ Receptors in Monkey Brain

[0040] We mapped the distribution of the receptors for Ang IV (AT₄receptors) in the Macaca fascicularis brain using in vitro receptorautoradiography in order to determine if the widespread and distinctdistribution of the receptors that are found in the guinea pig brain isalso found in primates. The binding sites were initially characterizedpharmacologically in competition studies on prefrontal cortical brainsections. These results are summarized in FIG. 1. Ang IV, Ang III andAng II competed for [¹²⁵I]Ang IV binding with IC₅₀s of 5 nM, 80 nM and730 nM respectively, while Ang II(1-7) was a weak competitor (IC₅₀ of 24mM). The AT₁ receptor antagonist, losartan (du Pont-Merck) and the AT₂receptor antagonist, PD 123319 (Parke-Davis), were inactive, even atconcentrations of 10 mM. These pharmacological properties are similar tothose previously described for the AT₄ receptor in bovine adrenal andguinea pig septal membranes, confirming that we were mapping thedistribution of the same receptor.

[0041] The distribution of the AT₄ receptor was remarkable, in that itsdistribution extended throughout several neural systems. This issummarized in Table 1. The most striking finding was the localization ofthis receptor in motor nuclei and motor-associated regions. Theseincluded the ventral horn spinal motor neurons, all cranial nerve motornuclei including the oculomotor, trochlear, facial and hypoglossalnuclei, and the dorsal motor nucleus of the vagus. Receptors were alsopresent in the vestibular, reticular and inferior olivary nuclei, thegranular layer of the cerebellum, and the Betz cells of the motorcortex. Moderate AT₄ receptor density was seen in all cerebellar nuclei,ventral thalamic nuclei and the substantia nigra pars compacta, with alower receptor density being observed in the caudate nucleus andputamen. The localization of the AT₄ receptor in all levels of the motorhierarchy in the central nervous system implies an important role forthe binding site in motor activity. TABLE 1 Localization andQuantitation of the AT₄ Receptor in the Macaca fascicularis Brain AT₄receptor density dpm/mm2 Region (mean ± SD) Caudate nucleus 48 ± 2Vertical limb of the diagonal band* 86 ± 3 Basal nucleus of Meynert* 81± 5 Granular layer of the dentate gyrus 117 ± 11 CA1 45 ± 4 CA3 41 ± 3Supraoptic retrochiasmatic nucleus* 93 ± 7 Ventral posteriorlateral/medial nuclei 35 ± 2 Red nucleus* 44 ± 2 Oculomotor nucleus* 44± 1 Pontine nuclei 50 ± 2 Lateral geniculate 52 ± 2 Mo5* 84 ± 3 Facialnucleus* 90 ± 4 Hypoglossal nucleus* 93 ± 8 Inferior olive  76 ± 10Granular layer of the cerebellum 126 ± 10 Molecular layer of thecerebellum 47 ± 6

[0042] In addition to the somatic motor nuclei and autonomicpreganglionic motor nuclei, abundant AT₄ receptors were also found inother cholinergic systems and their projections, including the nucleusbasalis of Meynert, vertical limb of the diagonal band and thehippocampus. Apart from being a neurotransmitter in motor neurons,acetylcholine is also implicated in cognition, since anti-cholinergicdrugs induce memory disorders and confusion; in Alzheimers's disease,neuronal loss occurs in the cholinergic-rich basal nucleus of Meynert.Ang IV has been shown by two independent studies to facilitate memoryretrieval in passive and conditioned avoidance tests (Braszko et al,1988; Wright et al, 1993), and, when administeredintracerebroventricularly, induces c-fos expression in the hippocampus(Roberts et al, 1995). Together with the presence of high densities ofAT₄ receptors in this region, these observations suggest that Ang IV mayplay an important role in the modulation of cognitive function.

[0043] AT₄ receptors were also observed in sensory regions, withmoderate levels in spinal trigeminal, gracile, cuneate and thalamicventral posterior nuclei, and in the somatosensory cortex. Whilereceptor density was low in sensory neurons when compared with thatobserved in motor and cognitive areas, the AT₄ receptor was locatedthroughout most sensory-associated areas, including the lamina II of thespinal cord, gracile, cuneate and spinal trigeminal nuclei, ventralposterior thalamic and lateral geniculate nuclei and the sensory cortex,suggesting a substantial involvement with sensory activity. Thisdistribution pattern has also been observed in the guinea pig and sheepbrain. As shown in Example 2, abundant AT₄ receptors were also observedin sheep dorsal root ganglia.

EXAMPLE 2 Mapping of Angiotensin AT₄ Receptors in Sheep Spinal Cord

[0044] We extended the localization of the AT₄ receptors to the sheepspinal cord, to investigate if the strong presence of the AT₄ receptorsin supraspinal motor and sensory regions persists in the spinal cord.

[0045] When the binding characteristics of [¹²⁵I]Ang IV were assessed inthe eighth cervical segment (C8) of the sheep spinal cord, we found thatthe affinities of the different unlabelled ligand in competing for thebinding were similar to those observed for the monkey brain.

[0046] In the sheep spinal cord, high densities of AT₄ receptors werefound in lamina IX in the ventral horns of all segments examined. At acellular level, the binding was found overlying the cytoplasm of lateraland medial motor neurons and in their processes, but binding was absentfrom the cell nuclei. Whilst a clearly defined function of the Ang IVbinding site is yet to be determined, the association with motoractivity is strengthened in view of its abundant localization in themotor neurones in the ventral horn of the spinal cord, in addition toits strong presence in supraspinal motor areas.

[0047] High densities of AT₄ receptors were also found in the lateraltip of lamina VII of all thoracic segments and lumbar segments L1 to L4,which corresponded with sympathetic preganglionic neurons in theintermediolateral cell column. However, binding was absent from L5 andL6 and from the sacral segments S1 and S2.

[0048] In the dorsal root ganglia associated with all spinal segments,high densities of AT₄ receptors were found in the cytoplasm of small andlarge cell bodies of the sensory neurons, but not in the satellitecells, nor in the endoganglionic connective tissue. In laminae I and II,the terminal fields of the dorsal root ganglia sensory afferents, only alow abundance of the receptor was noted in lamina II. Despite the lowlevels of AT₄ receptors in lamina II, their high abundance in the dorsalroot ganglia and their consistent but low levels in most supraspinalsensory areas suggest that AT₄ receptors may still play a role in theprocessing of sensory information.

[0049] Low levels of the AT₄ receptors were also found in the bloodvessels which extended radially to the pial surface, in the bloodvessels of the anterior and posterior fissures, and in the ependyma ofthe central canal. Ang IV has been reported to induce anendothelium-dependent dilation of rabbit pial arterioles, and in ratsAng IV reverses acute cerebral blood flow reduction after experimentalsubarachnoid haemorrhage.

[0050] Our localization studies suggest that AT₄ receptors are quitedistinct from the known angiotensin receptors—the AT_(1a), AT_(1b) andAT₂ receptors—in terms of their pharmacological specificity and theirpattern of distribution in the brain and spinal cord. Furthermore, thepattern of distribution of the AT₄ receptors suggests that they may beinvolved in the function of neurones involved in motor function, sensoryfunction and cholinergic systems, including cognition.

EXAMPLE 3 Characterization of Embryonic Chicken Ang IV and Ang IIBinding Sites

[0051] In order to characterize the pharmacology of the embryonicchicken AT₄ and Ang II receptors, chorioallantoic membranes (CAM) fromembryonic day 13 (E13) chickens were used. The membranes were removedand frozen in isopentane cooled to −40° C.

[0052] a) Characterization of the embryonic chicken Ang IV binding site

[0053] CAM were homogenized in 30 ml of a hypotonic buffer (50 mM Tris,pH 7.4, 5 mM EDTA) and then centrifuged for 10 min at 500 g and 4° C.The supernatant fraction was removed and centrifuged for 20 min at40,000 g and 4° C. The resulting pellet was rehomogenized in 2 ml ofhypotonic buffer, and the final volume of the homogenate was adjusted togive a protein concentration of 10 mg/ml, as determined by the Bioradprotein assay. The binding assay contained CAM (100 μg of protein), 0.14μCi of [¹²⁵]Ang IV (approximately 260 pM), and competing ligand, in atotal volume of 270 μl in a 50 mM Tris buffer, pH 7.4, containing 150 mMNaCl, 5 mM EDTA, 100 μM phenylmethylsulfonyl fluoride, 20 μM bestatinand 0.1% (w/v) bovine serum albumin. The binding system was incubated at37° C. for 2 h.

[0054] b) Characterization of the embryonic chicken Ang II binding site

[0055] CAM were prepared as described above with the followingexceptions. The isotonic buffer contained 50 mM Tris, pH 7.4 and 6.5 mMMgCl₂ and the hypotonic buffer contained 50 mM Tris, pH 7.4, 6.5 mMMgCl₂, 125 mM NaCl and 0.2% (w/v) bovine serum albumin. In addition, thepeptidase inhibitors, leupeptin, lisinopril, phosphoramidon, Plummer'sinhibitor and bestatin, each used at a 1 μM concentration and 1 mMbenzamidine and 2.5 mM phenanthroline, were included in both buffers.

[0056] In binding competition studies on E13 chicken CAM, [¹²⁵I]Ang IVbinding was strongly inhibited by Ang IV and Nle¹-AIV (IC₅₀s of 18 and43 nM respectively), whereas WSU-4042, Nle¹-Y-I-amide and Ang II wereweaker competitors with IC₅₀s of 5, 2.2 and 0.65 μM respectively, andlosartan and PD 123319, were inactive at concentrations up to 10 μM.[Sar¹Ile⁸]Ang II and CGP 42112 were effective at only competing for 50%of the sites, and then only at concentrations of 10 and 0.5 μMrespectively. These results are summarized in FIG. 2.

[0057] In studies of ¹²⁵I[Sar¹Ile⁸]Ang II binding to CAM, Ang II,[Sar¹Ile⁸]Ang II and CGP 42112 competed for binding with IC₅₀s of 100,13 and 180 nM respectively, whilst Ang IV, Nle¹-AIV and losartan werevery weak competitors (IC₅₀s of 50, 8 and 100 μM respectively). PD123319, WSU-4042 and Nle¹-Y-I-amide exhibited IC₅₀s greater than 100 μm.These results are shown in FIG. 3.

EXAMPLE 4 Effects of Ang IV on Neurite Outgrowth

[0058] The wide distribution of the AT₄ receptors in motor, sensory andcholinergic regions suggests important roles for this peptide in thecentral nervous system. However, a physiological action of Ang IV inneurons has yet to be clearly defined. Numerous neurotransmitters andneuropeptides have been associated with the regulation of neuronaldevelopment. For instance, acetylcholine inhibits neurite outgrowth fromembryonic chicken ciliary ganglion cells, sympathetic neurons, and rathippocampal neurons. Conversely, vasoactive intestinal peptidestimulates superior cervical ganglion branching and somatostatinincreases neuronal sprouting from Helisoma buccal ganglion neurons.

[0059] We determined whether Ang IV has a trophic role in the centralnervous system by examining its effects on neurite outgrowth fromcultured embryonic chicken sympathetic neurons.

[0060] Sympathetic ganglia from E11 chickens were dissociated usingtrypsin/Versene, and were cultured in 24 well plates in DMEM and Ham'sF12 medium which contained 1% (v/v) insulin-transferrin-selenium-Xgrowth supplement (Gibco BRL, Maryland USA), 100 mM putrescine, 1.67mg/ml prostaglandin F2α, 6.67 ng/ml progesterone, and 5 ng/ml nervegrowth factor (Sigma, Mo. USA). Neurons were allowed to adhere to thewells (approximately 2 h) before being given a 24 h treatment ofpeptides and/or their antagonists. Peptides and antagonists used wereadded to the cultures 0.5 h prior to either Ang IV or Ang II addition.Ang IV dose response curves were performed over the concentration range10⁻¹¹ to 10⁻⁵ M. Culture dishes were coated with 0.1 mg/ml polylysineand then given three washes with phosphate-buffered saline (PBS) beforebeing coated with 10 μg/ml laminin. Wells were washed with PBS beforebeing used for culture.

[0061] At the conclusion of the experiment, the culture medium wasremoved from the wells, the neurons were fixed with 2.5% glutaraldehydein PBS for 20 min and examined under a phase-contrast microscope,attached to an MD30 Plus image analysis software (Adelaide, Australia).The length of neurites (longer than 50 μm) of every neuron examined wasmeasured. A minimum of forty neurite measurements was taken pertreatment group, and each experimental treatment was performed at leastin triplicate.

[0062] At the conclusion of the experiment, the viability of the cellswere confirmed by exclusion of 0.1% aniline blue.

[0063] In cultures of embryonic (E11) chicken sympathetic neurons, AngIV inhibited neurite outgrowth in a dose-dependent manner, with athreshold at 10⁻¹¹ M, half maximal inhibition at 10⁻¹⁰ M and a maximaleffect at 10⁻⁹ M. Between 10⁻⁹ to 10⁻⁵ M, outgrowth was maximallyinhibited (P<0.05). These results are shown in FIG. 4. At 10⁻⁸ M Ang IV,the inhibition of neurite outgrowth was totally reversed by 1 μM of theAng IV analogues WSU-4042, Nle¹-Y-I-amide, and Nle¹-AIV. The effects ofthe analogues alone were not statistically different from controlvalues. In contrast to the Ang IV analogues, the Ang II antagonist,[Sar¹Ile⁸]Ang II, the AT₁ and AT₂ antagonists, losartan and PD 123319,and the Ang II partial agonist, CGP 42112, had no effect on the Ang IVresponse, as shown in FIG. 5.

[0064] At 10⁻⁸ M Ang II, neurite outgrowth was inhibited by 25%, whichwas highly significant. The Ang IV analogues completely reversed thiseffect, whilst the Ang II antagonists [Sar¹Ile⁸]Ang II, losartan, PD123319, and CGP 42112 were ineffective. This is illustrated in FIG. 6.

[0065] These studies suggest that the inhibition of neurite outgrowth byboth peptides is mediated by the AT₄ receptors, and supports a role forangiotensin IV in neurite modelling.

EXAMPLE 5 Effect of Angiotensin IV on Spinal Cord Damage

[0066] Glial fibrillary acid protein (GFAP)-positive astrocytes areinvolved with modelling neurite formation after damage to the spinalcord (Bovolenta et al, 1992). Injury-evoked plasticity is a similarsituation to that observed in the developing embryo (Schwartz, 1992). Inlight of our findings on the ability of spinal cord tissue to bind AngIV (Example 2), we tested the effect of spinal cord injury on Ang IVbinding. Surprisingly, we found a marked elevation of [¹²⁵I]Ang IVbinding in damaged spinal cord sections. This is illustrated in FIG. 7.

[0067] These results suggest that the AT₄ receptor may be a suitabletarget for alleviation of the effects of spinal cord injury.

EXAMPLE 6 Purification of an Endogenous Brain Peptide Which Binds to theAT₄ Receptor

[0068] The level of Ang IV in the brain is very low to undetectable (DJCampbell, personal communication). The widespread and characteristicdistribution of AT₄ receptors in the central nervous system suggeststhat there may be an as yet unidentified peptide ligand for thisreceptor. We therefore undertook a search for such a ligand, usingconventional protein chemistry purification techniques together with anAT₄ receptor assay system in order to detect and monitor substance(s) inextracts of sheep brain which compete for [¹²⁵I]Ang IV binding in thissystem.

[0069] a) ¹²⁵AT₄ Receptor Binding Assay

[0070] The binding of ¹²⁵I-Ang IV to bovine adrenal membranes was usedas an assay system to screen for AT₄ receptor binding activity in sheepcerebral cortex fractions. Bovine adrenal glands obtained from theabbatoir were diced into 1 mm×1 mm blocks, homogenized in 3 ml of ahypotonic buffer (50 mM Tris, 5 mM EDTA, pH 7.4) and then centrifugedfor 10 min at 500 g. The supernatant was removed and centrifuged for 20min at 40,000 g, and the resulting pellet was rehomogenized in 2 ml ofhypotonic buffer. Binding assay samples contained bovine adrenal (56 mgof protein as determined by the Biorad protein assay), 0.14 μCi of[¹²⁵I]Ang IV (approximately 260 pM), and 10 μl of test sample, in atotal volume of 270 μl in 50 mM Tris buffer, pH 7.4, containing 150 mMsodium chloride, 5 mM EDTA, 100 μM phenylmethylsulfonyl fluoride, 20 μMbestatin, and 0.1% (w/v) bovine serum albumin. The relative potency ofthe fractions in competing for ¹²⁵I-Ang IV binding was determined from astandard curve in which known amounts of unlabelled Ang IV were added(10⁻¹⁰ to 10⁻⁶ M). Fractions from each purification step were assayedfor their ability to compete for [¹²⁵I]Ang IV binding, with thoseexhibiting the highest activity undergoing the next purification step.

[0071] b) Purification Procedure

[0072] Sheep cerebral cortex was homogenized in 2 M acetic acid, (2 ml/gtissue), centrifuged, and the supernatant decanted. A preliminarypurification of the extract was performed using a column of preparativeC18 material (55-105 mm, Waters). The C18 eluent was lyophilized,reconstituted, and subjected to a series of chromatographic steps, inwhich fractions were assayed for Ang IV displacement activity. In brief,the chromatographic steps were: three successive reversed-phase HPLCsteps, using columns of varying pore size (Deltapak C18, 300°A, andNovapak C18) as well as changing ion-pairing agents, solvents andgradient elution conditions; this was followed by anion exchange, thencation exchange, with final purification on a microbore LC C8 column.The purified active peptide was sequenced using an Applied BiosystemsModel 470 A Protein Sequencer with an on-line Model 120A PTH Analyzer.

[0073] The sheep cerebral cortex yielded 1.9 nmoles of AT₄ receptorbinding activity per gram of wet weight after the first C18 Deltapakcolumn. Following the third Poly LC column (55° C.), Ang IV activitycoeluted with the major UV absorption peak, and the following peptidesequence was obtained from this peak:Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:1).

[0074] A search of protein database records revealed that this sequencecorresponded to the amino acid sequence 32-41 of the humane β, δ, γ andε globin chains and is known as LVV-haemorphin-7.

[0075] LVV-haemorphin-7 is a 10 amino acid peptide found in the brain,pituitary, hypothalamus and bone marrow which binds with high affinityto the angiotensin AT₄ receptor. The sheep peptide sequence is identicalto amino acids 30-39 of the sheep β_(A), β_(B), β_(C), and ε globinprecursors (Garner and Lingrel, 1989; Saban and King, 1994), and thissequence is conserved in many species, including human (see for exampleKarelin et al, 1994). In humans, there are 6 β-globin-like genes ε,γ^(A), γ^(C), δ, β and a pseudogene Ψβ, clustered on chromosome 11, andall encode the LVV-haemorphin-7 sequence (Karlsson and Nienhuis, 1985).This sequence is not present in any of the α globin family of genes.LVV-haemorphin-7 and some shorter sequences within this peptide haveopioid activity, and it appears that the sequence VVYP is required forthis activity (Karellin et al, 1994).

EXAMPLE 7 Properties of Synthetic LVV-haemorphin-7

[0076] A decapeptide with the sequence isolated above was synthesizedunder contract by Chiron Mimotopes, and its biochemical andpharmacological properties were characterized as follows:

[0077] a) HPLC

[0078] A preliminary high performance liquid chromatography (HPLC) runindicated that the synthetic peptide did not coelute with the fractionthat was sequenced. It appeared that the fraction might have beendegraded due to prolonged storage at 4° C. Mass spectrometry analysiswas carried out in order to determine whether this was the case. Thedata obtained from mass spectrometry analysis of the two active peaksproduced following prolonged storage of the original purified materialwere indeed consistent with degradation. The early eluting peak gave amass corresponding exactly to the loss of the phenylalanine residue fromthe carboxy terminus, whereas the second active peak gave a masscorresponding exactly to the loss of the amino terminal leucine residue.Furthermore, these data (given that all the mass readings wereunambiguous) strongly suggest that the active peptide is notpost-translationally modified, either in the peptide core or at theamino or carboxyl terminus.

[0079] b) Ligand Binding Studies

[0080] The pharmacological properties of the decapeptideLVV-haemorphin-7 in competing for the binding of ¹²⁵I-Ang IV in bovineadrenal membrane and sheep cerebellar cortical membranes weredetermined. Both LVV-haemorphin-7 and Ang IV were radioiodinated usingchloramine T, and separated on a C18 Sep-pak column using 0.5%trifluoroacetic acid in a 20-80% methanol gradient.

[0081] Bovine adrenal membranes or sheep cerebellar cortical membraneswere homogenized in 30 ml of a hypotonic buffer (50 mM Tris, 5 mM EDTA,pH 7.4), and then centrifuged for 10 min at 500 g. The supernatant wasremoved and centrifuged for 20 min at 40,000 g, and the resulting pelletwas rehomogenized in 2 ml of hypotonic buffer. Binding assays contained:

[0082] bovine adrenal (56 μg of protein) or sheep cerebellar membranes(26 μg of protein), as determined by the Biorad protein assay (Bradford,1976);

[0083]0.14 μCi of [¹²⁵I]Ang IV (approximately 260 pM), or 0.11 μCi of[¹²⁵I]LVV-haemorphin-7 (approximately 200 pM), and

[0084] competing ligand,

[0085] in a total volume of 270 μl in 50 mM Tris buffer, pH 7.4,containing 150 mM sodium chloride, 5 mM EDTA, 100 μMphenylmethylsulfonyl fluoride, 20 μM bestatin and 0.1% (w/v) bovineserum albumin.

[0086] The assay was incubated at 37° C. for 2 h.

[0087] In the bovine adrenal membranes, a range of concentrations ofunlabelled LVV-haemorphin-7 or Ang IV was added to the assay system inorder to determine the relative potencies of the two peptides in thisradioreceptor assay system. Both Ang IV and LW-haemorphin-7 displayedcomparable affinities in competing for the ¹²⁵I-Ang IV binding (approx.1-5 nM), with Ang IV exhibiting slightly higher affinity.

[0088] For competition studies in sheep cerebellar cortical membranes,dilutions of the unlabelled ligands, LVV-haemorphin-7, Ang IV, Ang II,Ang III and the non-specific opioid antagonist, naloxone, the Ang II AT₁antagonist, losartan, the Ang II AT₂ antagonist, PD 123319, and thesigma opioid and dopamine D₂ antagonist, haloperidol, were used atconcentrations ranging from 10⁻¹³ to 10⁻⁴ M. Quantitation of receptorbinding was calculated as the mean of two experiments.

[0089] In these studies, ¹²⁵I-LVV-haemorphin-7 binding to sheepcerebellar cortical membranes was competed for by LVV-haemorphin-7, AngIV, Ang III, and Ang II (IC₅₀s of 5.6 nM, 1 nM, 77 nM, and 1.6 μMrespectively). PD 123319 was a weak competitor (IC₅₀ of 46 μM), whilstlosartan, naloxone and haloperidol were ineffective (IC₅₀ greater than100 mM). These results are illustrated in FIG. 8. Similarly, [¹²⁵I]AngIV binding to cerebellar membranes was competed for by Ang IV,LVV-haemorphin-7, Ang III, and Ang II with IC₅₀S of 1.13 nM, 2 nM, 6.9nM and 2 μM respectively, whilst PD 123319, losartan, naloxone andhaloperidol were inactive at 10 μM. These results are illustrated inFIG. 9.

[0090] c) Binding of ¹²⁵1I-LVV-haemorphin-7 to Sheep Brain

[0091] Sheep hindbrain sections were used to compare the distribution of¹²⁵I-LVV-haemorphin-7 binding and AT₄ receptor sites. Sections at 10 μmthickness were equilibrated to 22° C. (30 min), and then preincubatedfor 30 min in an isotonic buffer containing 50 mM Tris, 150 mM sodiumchloride, 5 mM EDTA, 100 μM phenylmethylsulfonyl fluoride, 20 μMbestatin and 0.1% bovine serum albumin, pH 7.4, before a further 2 hincubation in the same buffer containing 2.84 μCi of[¹²⁵I]LVV-haemorphin-7 or [125I]Ang IV (approximately 140 pM). Thebinding of the radioligands was cross-displaced with either 1 μMunlabelled LVV-haemorphin-7 or Ang IV. After incubation, the sectionswere given three 2 min washes in buffer at 4° C., and exposed to X-rayfilm for 14 to 28 d.

[0092] [¹²⁵I]LVV-haemorphin-7 and [¹²⁵I]Ang IV exhibited an identicalbinding pattern in the sheep hindbrain. Binding was localized to themotor-associated areas, the granular layer of the cerebellum, theinferior olive, hypoglossal and lateral reticular nuclei, to theautonomic regions, the dorsal motor nucleus of the vagus and the nucleusambiguus, and to the sensory regions, the external cuneate and spinaltrigeminal nuclei. The binding of both radioligands was displaced by a 1μM concentration of either unlabelled Ang IV or LVV-haemorphin-7,indicating that not only are the two binding sites distributed in thesame brain regions, but that the two radioligands are actually bindingto the same sites.

EXAMPLE 8 Isolation of Potential LVV-Haemorphin-7 Precursor Clones

[0093] It is not known whether LVV-haemorphin-7 is synthesized in thebrain, or whether it is derived from the breakdown of haemoglobin.Demonstration of LVV-haemorphin-7 precursor mRNA in the brain wouldprovide evidence for the former. Possible methods to demonstrate thatLVV-haemorphin-7 precursor mRNA is present in the brain include:

[0094] (a) isolation of specific cDNA clones from a brain cDNA library;

[0095] (b) detection of the mRNA in the brain by RT-PCR;

[0096] (c) detection of LVV-haemorphin-7 precursor MRNA by in situhybridization histochemistry; and

[0097] (d) demonstration of the MRNA in brain specific cell cultures.

[0098] It has previously been reported that α- and β-globin mRNAs areexpressed in mouse brain, as demonstrated by Northern analysis(ohyagi,Y., et al, 1994).

[0099] Each of these approaches has specific advantages. In situhybridization histochemistry and detection of the mRNA in brain specificcell cultures would provide evidence for synthesis in the brain.Isolation of clones and the reverse transcription polymerase chainreaction (RT-PCR) detection of mRNA would show the presence of mRNA inthe brain, but contamination by reticulocytes cannot be excluded.However, isolation of cDNA clones provides considerable informationabout the structure of the precursor. The precursor of LVV-haemorphin-7may be a member of the β-globin family, eg β^(A) etc, or analternatively spliced globin, or it may be a previously unknownnon-globin peptide.

[0100] To isolate potential clones that code for the precursor of theLVV-haemorphin-7 peptide, we have screened a rat brain cDNA libraryusing an oligonucleotide based on the LVV-haemorphin-7 sequence.

[0101] Oligonucleotide Design

[0102] A number of oligonucleotides have been designed, as illustratedin FIG. 10. Oligonucleotide H170 (SEQ ID NO:2) was designed tocorrespond to the region of the sheep β-globin gene encoding theLVV-haemorphin-7 sequence. This probe was used for screening thelibrary, and also as the sense oligonucleotide in PCR. OligonucleotideH173 (SEQ ID NO:5) was designed as the antisense primer for use in PCR.PCR with H170/H173 spans intron 2, and will generate a 255 bp fragmentwith cDNA as the template and a 1098 bp fragment with genomic DNA.Oligonucleotide H172 (SEQ ID NO:4) can be used as an internal probe forH170/H173 PCR products. Oligonucleotide H172 and H173 (SEQ ID NO:4, 5)are antisense probe corresponding to exon 2 and 3 of the sheep β-globingene, and were used for in situ hybridization histochemistry.

[0103] Detection of β-Globin Like Sequences in Brain by Polymerase ChainReaction (PCR)

[0104] RNA was isolated from sheep cerebellar and cerebral cortices,heart and liver. The RNA (20 μg) was reverse transcribed in a 25 μlreaction containing 100 mM KCl, 50 mM Tris-HCl (pH 8.4), 6 mM MgCl₂, 10mM dithiothreitol, 500 μM dNTPs (Progen), 12μg/ml random hexamers(Boehringer Mannheim), 40 units RNasin (Progen), and AMV reversetranscriptase (Boehringer Mannheim, 25 units) at 42° C. for 1 h. Analiquot of the reverse transcription reaction (10% ) was used in thepolymerase chain reaction. The primers used for amplification of theβ-globin mRNA were sense H170 and antisense H173 (see FIG. 10). PCR wasperformed in a reaction containing: 10 mM Tris-HCl (pH 8.3), 50 mM KCl,400 μM dNTPs, Taq Polymerase (Bresatec, 2.5 units), 3 μM MgCl₂,and eachprimer at 400 nM. Denaturation, annealing and extension were carried outat 94° C., 60° C. and 72° C. for 1 min each for 40 cycles, followed by afinal extension at 72° C. for 10 min.

[0105] The PCR products were separated on an agarose gel, transferred toHybond N+, and Southern analysis using an internal oligonucleotide(H172) was performed to confirm that the products were derived fromglobin precursors. Specific bands of the expected size of 255 bp weredetected in all four tissues examined, as shown in FIG. 11.

[0106] Screening a Rat cDNA Library for β-Globin Like Sequences

[0107] An oligonucleotide corresponding to the nucleotide sequence ofthe LVV-haemorphin-7 region of the sheep β-globin (H170) was used toscreen a rat brain cDNA library (Stratagene Cat No: 936515,Sprague-Dawley, whole brain). Approximately 8×10⁵ clones were plated,and plaque lifts taken using standard methods (eg Maniatis et al:Molecular Cloning). The filters were prehybridzed in Rapid-Hyb(Amersham) for 1 hr at 42° C., then the 5′ end labelled H170 was addedfor 2 hr. The filters were then washed 3 times at 42° C. in 2× SSC/0.1%SDS. The filters were autoradiographed for 4 days using Biomax film andan intensifying screen. A total of 24 putative positives was isolated.The positives were eluted in PSB.

[0108] The positives were then further characterized using a PCR basedmethod. PCR was performed using oligonucleotide H170 as the 5′ primerand H173 as the 3′ primer. A PCR product derived from H170/H173 willspan an intron in the sheep β-globin gene, and will generate a 1098 bpfragment.

[0109] An aliquot of the eluted λ clone was boiled for 5 min, thenchilled on ice. This was used as template DNA in a PCR reactioncontaining 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 400 μM dNTPs, TaqPolymerase (Bresatec, 2.5 units), 3 μM MgCl₂,and each primer at 400 nM.Denaturation, annealing and extension were carried out 94° C., 60° C.and 72° C. for 1 min each for 30 cycles, followed by a final extensionat 72° C. for 10 min. PCR products were analysed by electrophoresis on a1.4% agarose gel.

[0110] The H170 positive/PCR negative clones were stored for furthercharacterization. It is considered that they may be either non-globinprecursors, alternatively spliced precursors or fragments of globinclones.

[0111] Sequencing Rat β-globin Clones

[0112] The 6 positives selected by PCR were plaque purified, andsubjected to plasmid excision according to the manufacturersinstructions. The insert sizes were determined by separate restrictionmapping with the enzymes EcoRI and PvuII. Clones EX, FX, LX, RX and TXcontain inserts of approx 500 bp. Clone DX was the longest, andcontained an insert of approximately 2500 bp. Southern analysis of theclones using an internal oligonucleotide (H172) confirmed that theseclones were derived from globin precursors.

[0113] These plasmids were sequenced using the Pharmacia T7 sequencingkit. Sequencing of clones EX, FX and LX, using the universal primer,showed sequence homology to the 3′ untranslated region of β-globin.Clones RX and TX when sequenced with the universal primer, and clone DXwhen sequenced with the reverse primer, showed sequence homology to the5′ end of the β-globin gene, including the initiation codon ATG.

[0114] Clone DX was subjected to nested deletion analysis to generatemore templates for sequencing. This clone contained the β-globinsequence, and approximately 1.8 kb of sequence which was not homologousto the globin cluster, and may be the result of two inserts in the oneclone.

[0115] Complete sequencing of clone EX showed that the clone wasidentical to rat β^(A)-globin (Genbank accession No: X16417), as shownin FIG. 12. FIG. 13 shows the nuclectide sequence and derived amino acidsequence of clone EX, indicating the putative LVV-haemorphin-7 region.

EXAMPLE 9 In situ Hybridization Histochemistry

[0116] The distribution of mRNA encoding LVV-haemorphin-7 and itsprecursor peptide is being investigated using a range ofoligonucleotides for the different regions of the β-globin gene,including the C-terminal of exon 2 (H172 of FIG. 13) and the N-terminalregions of exon 3 (H173). The antisense (initially H172, H173)oligonucleotides were 3′ end labelled with a ³⁵S-dATP using terminald-transferase and purified on a Nensorb column. Sheep brain sectionswere then hybridized with 7.5×10⁵ cpm of labelled probe in a 75 μl totalvolume of 50% formamide, 4× SSC, 1× Denhardt's solution, 2% sarcosyl, 20mM Na₂PO4 buffer (pH 7), 10% dextran sulphate, 50 μg/ml herring spermDNA and 0.2 mM dithiothreitol. After a 16 h hybridization period, thesections were washed four times in 1× SSC, rinsed in distilled water anddehydrated through increasing ethanol and exposed to Hyperfilm β-max.Preliminary experiments using oligonucleotides H172 and H173 detectedβ-globin MRNA in the inferior colliculus and nucleus of the spinaltrigeminal. Further in situ hybridization histochemical studies involvethe use of additional antisense and sense synthetic oligonucleotidesfrom different regions of the β-globin sequence to confirm our findingof β-globin MRNA in brain nuclei. The distribution of β-globin mRNA isthen compared to our autoradiographic localization of the AT₄ receptorsin order further to lucidate roles for this novel peptide system.

EXAMPLE 10 Radioimmunoassay and Immunohistochemical Detection ofLVV-haemorphin-7

[0117] Two sheep were immunized with the LVV-haemorphin-7 sequenceconjugated to diphtheria toxoid and both antisera and affinity purifiedantisera with adequate titre to set up radioimmunoassays forLVV-haemorphin-7 have been obtained. The radioimmunoassay, which is ofconventional type, is used to determine the concentration ofLVV-haemorphin-7 in different tissues or in specific regions within atissue, in order to provide us with further information as to otherpossible physiological actions of the decapeptide.

[0118] The antisera are also used immunohistochemically to determine thetissue distribution of LVV-haemorphin-7, particularly in the brain.Guinea pigs are perfused intracardially with 4% paraformaldehyde inphosphate-buffered saline solution, the tissues dissected out andimmersed in a 20% sucrose solution overnight. The tissues are thenfrozen, 5-10 micron sections cut, and endogenous peroxidase blocked by a30 min incubation in 0.5% hydrogen peroxidase in methanol prior to anovernight incubation with the primary antibody in phosphate-bufferedsaline containing 3% normal goat serum. After a few washes in phosphatebuffered saline, the sections are incubated with the secondaryanti-sheep antibody, and detected using thestreptavidin-biotin/horseradish peroxidase complex system (Vectastain).The detection of LVV-haemorphin-7 in neurones provides further supportthat the decapeptide is synthesized within neurones, and thereby mayfunction as a neuropeptide, since we have already shown that itsreceptor occurs in neurones. Immunohistochemistry is also performed atthe electron microscopic level in order to evaluate the subcellulardistribution of the peptide, in particular whether it occurs inintracellular storage granules.

[0119] The radioimmunoassay for LVV-haemorphin-7 is also employed toinvestigate the secretion of the peptide from neural tissue. Slicesprepared from brain regions found to be rich in LVV-haemorphin-7immunoreactivity are incubated in Krebs Ringer Bicarbonate buffer at 37°C., and the effects of depolarization by high K⁺ medium and varioussecretagogues are evaluated to test whether the peptide is secreted fromneurones. Similar experiments are carried out on cultured neuronal celllines which are found to contain the peptide. Radioimmunoassays of bodyfluids including plasma and cerebrospinal fluid are used to determinelevels of the peptide in these fluids under normal and pathologicalconditions.

[0120] In addition, the subcellular distribution of the peptide isevaluated by radioimmunassay of subcellular fractions from nervoustissues, including synaptosomes, in order to evaluate if the peptide isstored in subcellular granules, as occurs for other secretedneuropeptides.

EXAMPLE 11 Effect of LVV-haemorphin-7 in Passive Avoidance ConditioningTrials

[0121] Angiotensin IV has been shown to improve memory retention andretrieval in a passive avoidance task (Braszko et al, 1988, Wright etal, 1993), an effect which was mediated via the AT₄ receptor.Scopolamine , a muscarinic receptor antagonist, has been used to induceamnesia. It has been reported that a more stable analogue of angiotensinIV, WSU 2088, reversed the disruption in learning in a passive avoidancetask induced by scopolamine. The effects of LVV-haemorphin-7 on theconditioned passive avoidance task in untreated and scopolamine-treatedrats were tested.

[0122] Rats were surgically implanted with intracerebroventricularcannulae and handled daily. On the conditioning day, each animal washabituated to the dark compartment of a passive avoidance conditioningapparatus for 5 min with the guillotine door closed. The animal was thenreturned to its home cage for 5 min and then placed in the lightcompartment with the guillotine door opened. Latency to enter the darkcompartment with all four feet was measured in seconds. These trialswere repeated with 5 min in the home cage between trials until the ratentered the dark side within 20 seconds. Before the final trial onconditioning day, the rats were randomly divided into four groups: (a)saline followed by saline (b) saline followed by 1.0 nmolLVV-haemorphin-7 in (c) 70 nmol scopolamine followed by saline (d) 70nmol scopolamine followed by 1.0 nmol LVV-haemorphin-7, all administeredin a volume of 2.5 μl intracerebroventricularly 30 min and 5 min beforethe final trial respectively. On the last trial, the guillotine door wasclosed and the animals received one low-level shock (0.2 mA) for 1.5seconds via the grid floor. The animals were then returned to their homecages for 24 hours before being tested once daily for the next four daysand the latency periods to reenter the dark compartment were measured.Results are shown in FIG. 14.

[0123] In this passive avoidance paradigm, the control animals whichreceived successful conditioning displayed high latencies in enteringthe darkened compartment, whereas rats treated with scopolaminedisplayed learning and memory deficits, as indicated by much lowerlatencies in entering the dark compartment. The mean latencies to enterthe dark compartment of rats which received LVV-haemorphin-7 afterscopolamine were not significantly different from those of the controlrats, indicating that in these rats LVV-haemorphin-7 completely reversedthe scopolamine-induced amnesia. However, the rats which receivedLVV-haemorphin-7 alone performed worse than the scopolamine-treatedrats.

[0124] These results indicate that LVV-haemorphin-7 successfullycounteracts the memory disruption induced by scopolamine treatment.However, administration of the peptide alone was detrimental tolearning, which may be due to overstimulation of the neuronal systembecause of the high dose used.

[0125] Effective doses of LVV-haemorphin-7 are determined by conductingdose-response studies with LVV-haemorphin-7 and observing the effects onlearning a passive avoidance task in the animals, including those withscopolamine-induced amnesia. Similar studies are also used to determineif the memory disruption caused by LVV-haemorphin-7 is due toexcessively high doses of the peptide.

EXAMPLE 12 Effect of LVV-haemorphin-7 in the Water Maze AcquisitionTrials

[0126] The circular water maze (Morris water maze) consists of acircular tank containing water which has been rendered opaque, with ahidden platform underneath the surface of the water. Scopolamine blocksthe trial-to-trial decrease in latency of this task, and this effectappears to be due to impairment of short-term memory. The effect ofLVV-haemorphin-7 on the scopolamine-induced amnesia in this task wasinvestigated.

[0127] Rats were surgically implanted with intracerebroventricularcannulae and handled daily. On the day of the trial, the rats wereintroduced into the water maze from different starting positionsequidistant from the escape platform . The time taken for each rat toreach the platform was noted. There were four consecutive trials foreach animal on each day, with a 60 second rest period between trials.The mean latency period before the animal reached the platform wasplotted, and is shown in FIG. 15. On days 1 and 2 of the trial(non-spatial), none of the animals received any drug treatment. Althoughthe scopolamine group displayed increased latency on day 1, the latencyon day 2 decreased to control level. The rats were then randomly dividedinto 3 groups:

[0128] (a) the saline control,

[0129] (b) 70 nmol scopolamine in 2.5 μl, and

[0130] (c) 70 nmol scopolamine followed by 1.0 nmol LVV-haemorphin-7,and were subjected to 5 days of testing. Upon intracerebroventriculartreatment with scopolamine 30 min prior to testing, the rats displayedsignificantly increased latencies in finding the platform, demonstratingdeficits in learning. In rats treated with LVV-haemorphin-7 25 min afterscopolamine, the scopolamine-induced latency in finding the platform wastotally reversed, and these rats were indistinguishable from the controlgroup. Withdrawal of treatment on day 8 brought the latency ofscopolamine-treated group back to control levels, indicating that thescopolamine-induced amnesia is reversible.

EXAMPLE 13 Effect of LVV-Haemorphin-7 on Acetylcholine Release in RatHippocampus

[0131] Acetylcholine is thought to be the major transmitter involved inthe processing of cognitive function, since anti-cholinergic drugsinduce memory deficit and confusion. In Alzheimer's disease, neuronalloss has been reported in cholinergic-rich areas, particularly in theseptohippocampal pathway. Angiotensin AT₄ receptors were found in highabundance in the basal nucleus of Meynert, in the CA2 and dentate gyrusof the hippocampus, and in somatic and autonomic preganglionicmotoneurones of the monkey brain. This pattern of receptor distributionclosely resembles that of cholinergic neurones, and suggests that theAT₄ receptors may be associated with cholinergic pathways centrally.Moreover, as shown in Example 12 LVV-haemorphin-7 can reverse thelearning deficit induced by scopolamine (a muscarinic receptorantagonist). We therefore postulate that LVV-haemorphin-7 can modulateacetylcholine release from the septohippocampal neurones via the AT₄receptors.

[0132] Rats are anaesthetized with sodium pentobarbitone, andstereotaxically implanted with intracerebral guide cannulae either inthe dorsal hippocampus (coordinates 3.8 mm caudal to bregma, 2.5 mmlateral to midline, and 3.0 mm ventral to the surface of the skull) orventral hippocampus (coordinates 5.3 mm caudal to bregma, 5.4 mm lateralto midline, and 6.5 mm ventral to surface of the skull). The guidecannulae are secured with dental cement anchored to three screws in theskull. Dummy probes are then inserted into the guide cannulae to preventblockade of the cannulae. The rats are allowed to recover for 5-7 days.On the day of the experiment, a microdialysis probe, with a 3 mmdialysis membrane, is inserted through the guide cannula and perfusedwith artificial cerebrospinal fluid (148 mM NaCl, 3 mM KCl, 1.4 mM CaCl,0.8 mM MgCl, 1.3 mM NaH₂PO₄, 0.2 mM Na₂HPO₄, pH 7.4) at a flow rate of2.0 μl/min. Neostigmine (1.0 μM) is added to the artificialcerebrospinal fluid to facilitate recovery of acetylcholine. Four 20-minbaseline samples are collected 1 h after probe insertion, followed byfour 20-min samples during the experimental period when LVV-haemorphin-7(1 μmol dissoolved in artificial cerebrospinal fluid and 1 μMneostigmine) is perfused through the probe. During the recovery period,the peptide perfusion is withdrawn and four 20-min samples arecollected.

[0133] Acetylcholine in the dialysates is measured by HPLC withelectrochemical detection. Acetylcholine and choline are separated on a10 cm polymer-based analytical column, and then converted to hydrogenperoxide and betaine by an immobilized enzyme reactor(acetylcholinesterase and choline oxidase) coupled to the analyticalcolumn. The mobile phase consists of 35 mM sodium phosphate at pH 8.5supplemented with the antibacterial reagent Kathoon CG.

EXAMPLE 14 Detection of β-globin Sequences in Different Neuronal CellLines by RT-PCR

[0134] Total RNA is isolated from the following cell lines:

[0135] (a) NG 108 rat glioma-neuroblastoma hybrid,

[0136] (b) SKNMC human neuroblastoma, and

[0137] (c) PC 12W rat pheochromocytoma. The total RNA is prepared asfollows: 10⁷ cells are homogenized in 4 ml of 4M guanidine thiocyanate,25 mM sodium citrate and 0.05% sodium dodecyl sulphate followed bysequential addition of 0.4 ml of 2 M sodium acetate pH 4.0, 4 ml watersaturated phenol, and 0.8 ml of chloroform-isoamyl alcohol. Thehomogenate is mixed and cooled on ice for 15 min followed bycentrifugation at 2000 g for 15 min. The aqueous phase is removed andsubjected to 2 phenol-chloroform extractions before RNA is precipitatedby the addition of isopropanol.

[0138] The mRNAs are then subjected to RT-PCR. cDNA is synthesized fromapproximately 20 μg of total RNA, using reverse transcriptase and randomhexamers. Ten percent of the cDNA product was amplified by PCR through40 cycles, with each cycle consisting of denaturation at 94° C. for 1min, annealing of primers at 60° C. for 1 min and primer extension at72° C. for 1 min, followed by a final 10 min incubation ac 72° C. Theprimers used were 5′ CTGGTTGTCTACCCCTGGACTCAGAG3′ (SEQ ID NO:2), and 5′CAGCACAACCACTAGCACATTGCC3′ (SEQ ID NO:5), which corresponded with highhomology to sheep β,δ, ε globin chains and flanked a 255 bp cDNAfragment. The sense primer spans the nucleotide sequence which coded forLVV-haemorphin-7, and the antisense primer spans the second intron ofthe globin gene, to enable cDNA to be distinguished from contaminatinggenomic DNA. The PCR products are transferred to a Hybond N+ membrane bydownward Southern blotting in 0.4 M NaOH. The membrane is hybridized at42° C. in 5× SSC, 5× Denhardt's solution and 0.5% sodium dodecylsulphate, with a ³²p end-labelled oligonucleotide 5′CTCAGGATCCACATGCAGCTTATCACAG3′ (SEQ ID NO:3), which is internal to theprimers used for PCR and binds to β,δ and ε globin chains. After 12 h ofhybridization, the filter is washed at 42° C. in a buffer with a finalstringency of 0.5× SSC and 0.1% sodium dodecyl sulphate.

[0139] We have mapped the distribution of AT₄ receptors in the brain ofMacaca fascicularis and sheep spinal cord. The receptor has a strikingand unique distribution, including motor- and sensory-associated regionsand pathways and cholinergic cell bodies, including all motor nuclei inthe brain stem and spinal cord. We have demonstrated that Ang IVinhibits neurite outgrowth in cultured embryonic chicken neurones, andthat this peptide may therefore have a role in growth and development ofthe central and peripheral nervous systems.

[0140] We have purified an endogenous brain peptide which binds to theAT₄ receptor with high affinity. This decapeptide is 100% identical tothe internal amino-acid sequence 30-39 of sheep β-globin. The presenceof this β-globin-like sequence was demonstrated in sheep brain and othertissues using PCR. Screening of a rat brain cDNA library led to theisolation of a clone identical in sequence to rat β^(A)-globin.

[0141] We have demonstrated the presence of β-globin mRNA in braintissue and isolated a β-globin cDNA clone from a rat brain library.These data suggest that LVV-haemorphin-7 is derived from β-globinprecursors synthesized in the brain, although contamination byreticulocytes cannot be excluded. All of the cDNA clones sequencedcorrespond to the sequence encoding rat β^(A)-globin. The ratLVV-haemorphin-7 peptide sequence has a conservative substitution atposition 10, with a tyrosine replacing a phenylalanine.

[0142] It therefore appears that a peptide corresponding to the sequenceof the bovine LVV-haemorphin-7 exists in brain, and is derived fromβ-globin as precursor. The peptide is almost certainly an endogenousligand for abundant brain AT₄ receptors, and may therefore exert a rangeof actions on defined motor sensory and cholinergic neurones.

[0143] We have shown that LVV-haemorphin-7 reverses thememory-disruptive effects of scopolamine in both passive avoidanceconditioning trials and in water maze acquisition trials. However,administration of high doses of the peptide may be detrimental tolearning due to overstimulation of the neuronal system.

[0144] In a wider context, our findings suggest that β-globin may be aprecursor of a range of neuroactive peptides generated in the centralnervous system by specific cleavage enzymes to interact with a range ofreceptors.

[0145] It will be apparent to the person skilled in the art that whilethe invention has been described in some detail for the purposes ofclarity and understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this invention.

[0146] References cited herein are listed on the following pages, andare incorporated by this reference.

REFERENCES

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[0148] 2. Braszko, J. J., Kupryszewski, G., Witczuk, B. and Wisniewski,K. Neurosci., 1988 27 777-783.

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[0150] 4. Haberl, R. L., Decker, P. J. and Einhaupl, K. M., Circ. Res.,1991 68 1621-1627.

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[0152] 6. Karelin, A. A., Philippova, M. M., Karelina, E. V. and Ivanov,V. T. Biochem. Biophys. Res. Comm., 1994 202 410-415

[0153] 7. Miller-Wing, A. V., Hanesworth, J. M., Sardinia, M. F., Hall,K. L., Wright, J. W., Speth, R. C., Grove, K. L. and Harding, J. W. J.Pharmacol. Exp. Ther. 266 (1993) 1718-1726.

[0154] 8. Moeller, I., Chai, S. Y., Oldfield, B. J., McKinley, M. J.,Casley, D. and Mendelsohn, F. A. O. Brain Res., 1995 701 301-306.

[0155] 9. Moeller, J., Paxinos, G, Mendelsohn, F. A. O., Aldred, G. P.,Casley, D and Chai, S. y., Brain Ros, 1996 712 307-324.

[0156] 10. Ohyagi, Y., Yamada, T. and Goto, I. Brain Res., 1994 635323-327

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[0158] 12. Saban, J. and King, D. Biochim. Biophys. Acta., 1994 121887-90

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[0160] 14. Swanson, G. N., Hanesworth, J. M., Sardinia, M. F., Coleman,J. K. M., Wright, J. W., Hall, K. L., Miller-Wing, A. V., Stobb, J. W.,Cook, V. I., Harding, E. C. and Harding, J. W. Reg. Peptides, 1992 40409-419.

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[0162] 16. Wong, P. C., Hart, S. D., Zaspel, A. M., Chiu, A. T.,Ardecky, R. J., Smith, R. D. and Timmermans, P. B. J. Pharmacol. Exp.Ther. 255 (1990) 584-592.

[0163] 17. Wright, J. W., Miller-Wing, A. V., Shaffer, M. J., Higginson,C., Wright, D. E., Hanesworth, J. M. and Harding, J. W. Brain Res.Bull., 1993 32 497-502.

[0164] 18. Wright, J. W., Krebs, L. T., Stubb, J. W. and Harding, J. W.Neuroendocrinology, 1995 16 23-52

1 10 1 10 PRT Unknown Organism Description of Unknown Organism Betaglobin precursor 1 Leu Val Val Tyr Pro Trp Thr Gln Arg Phe 1 5 10 2 26DNA Artificial Sequence Description of Artificial Sequence Primer 2ctggttgtct acccctggac tcagag 26 3 26 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 3 ctctgagtcc aggggtagac aaccag 26 4 28 DNAArtificial Sequence Description of Artificial Sequence Primer 4ctcaggatcc acatgcagct tatcacag 28 5 24 DNA Artificial SequenceDescription of Artificial Sequence Primer 5 cagcacaacc actagcacat tgcc24 6 613 DNA Rattus sp. 6 cacaaactca gaaacagaca ccatggtgca cctgactgatgctgagaagg ctgctgttaa 60 tggcctgtgg ggaaaggtga accctgatga tgttggtggcgaggccctgg gcaggctgct 120 ggttgtctac ccttggaccc agaggtactt tgatagctttggggacctgt cctctgcctc 180 tgctatcatg ggtaacccta aggtgaaggc ccatggcaagaaggtgataa acgccttcaa 240 tgatggcctg aaacacttgg acaacctcaa gggcacctttgctcatctga gtgaactcca 300 ctgtgacaag ctgcatgtgg atcctgagaa cttcaggctcctgggcaata tgattgtgat 360 tgtgttgggc caccacctgg gcaaggaatt caccccctgtgcacaggctg ccttccagaa 420 ggtggtggct ggagtggcca gtgccctggc tcacaagtaccactaaacct cttttcctgc 480 tcttgtcttt gtgcaatggt caattgttcc caagagagcatctgtcagtt gttgtcaaaa 540 tgacaaagac ctttgaaaat ctgtcctact aataaaaggcatttactttc actgcaaaaa 600 aaaaaaaaaa aaa 613 7 613 DNA Rattus sp. CDS(23)..(463) 7 cacaaactca gaaacagaca cc atg gtg cac ctg act gat gct gagaag gct 52 Met Val His Leu Thr Asp Ala Glu Lys Ala 1 5 10 gct gtt aatggc ctg tgg gga aag gtg aac cct gat gat gtt ggt ggc 100 Ala Val Asn GlyLeu Trp Gly Lys Val Asn Pro Asp Asp Val Gly Gly 15 20 25 gag gcc ctg ggcagg ctg ctg gtt gtc tac cct tgg acc cag agg tac 148 Glu Ala Leu Gly ArgLeu Leu Val Val Tyr Pro Trp Thr Gln Arg Tyr 30 35 40 ttt gat agc ttt ggggac ctg tcc tct gcc tct gct atc atg ggt aac 196 Phe Asp Ser Phe Gly AspLeu Ser Ser Ala Ser Ala Ile Met Gly Asn 45 50 55 cct aag gtg aag gcc catggc aag aag gtg ata aac gcc ttc aat gat 244 Pro Lys Val Lys Ala His GlyLys Lys Val Ile Asn Ala Phe Asn Asp 60 65 70 ggc ctg aaa cac ttg gac aacctc aag ggc acc ttt gct cat ctg agt 292 Gly Leu Lys His Leu Asp Asn LeuLys Gly Thr Phe Ala His Leu Ser 75 80 85 90 gaa ctc cac tgt gac aag ctgcat gtg gat cct gag aac ttc agg ctc 340 Glu Leu His Cys Asp Lys Leu HisVal Asp Pro Glu Asn Phe Arg Leu 95 100 105 ctg ggc aat atg att gtg attgtg ttg ggc cac cac ctg ggc aag gaa 388 Leu Gly Asn Met Ile Val Ile ValLeu Gly His His Leu Gly Lys Glu 110 115 120 ttc acc ccc tgt gca cag gctgcc ttc cag aag gtg gtg gct gga gtg 436 Phe Thr Pro Cys Ala Gln Ala AlaPhe Gln Lys Val Val Ala Gly Val 125 130 135 gcc agt gcc ctg gct cac aagtac cac taaacctctt ttcctgctct 483 Ala Ser Ala Leu Ala His Lys Tyr His140 145 tgtctttgtg caatggtcaa ttgttcccaa gagagcatct gtcagttgttgtcaaaatga 543 caaagacctt tgaaaatctg tcctactaat aaaaggcatt tactttcactgcaaaaaaaa 603 aaaaaaaaaa 613 8 147 PRT Rattus sp. 8 Met Val His Leu ThrAsp Ala Glu Lys Ala Ala Val Asn Gly Leu Trp 1 5 10 15 Gly Lys Val AsnPro Asp Asp Val Gly Gly Glu Ala Leu Gly Arg Leu 20 25 30 Leu Val Val TyrPro Trp Thr Gln Arg Tyr Phe Asp Ser Phe Gly Asp 35 40 45 Leu Ser Ser AlaSer Ala Ile Met Gly Asn Pro Lys Val Lys Ala His 50 55 60 Gly Lys Lys ValIle Asn Ala Phe Asn Asp Gly Leu Lys His Leu Asp 65 70 75 80 Asn Leu LysGly Thr Phe Ala His Leu Ser Glu Leu His Cys Asp Lys 85 90 95 Leu His ValAsp Pro Glu Asn Phe Arg Leu Leu Gly Asn Met Ile Val 100 105 110 Ile ValLeu Gly His His Leu Gly Lys Glu Phe Thr Pro Cys Ala Gln 115 120 125 AlaAla Phe Gln Lys Val Val Ala Gly Val Ala Ser Ala Leu Ala His 130 135 140Lys Tyr His 145 9 620 DNA Unknown Organism Description of UnknownOrganism RNBGLO 9 tgcttctgac atagttgtgt tgactcacaa actcagaaac agacaccatggtgcacctga 60 ctgatgctga gaaggctgct gttaatggcc tgtggggaaa ggtgaaccctgatgatgttg 120 gtggcgaggc cctgggcagg ctgctggttg tctacccttg gacccagaggtactttgata 180 gctttgggga cctgtcctct gcctctgcta tcatgggtaa ccctaaggtgaaggcccatg 240 gcaagaaggt gataaacgcc ttcaatgatg gcctgaaaca cttggacaacctcaagggca 300 cctttgctca tctgagtgaa ctccactgtg acaagctgca tgtggatcctgagaacttca 360 ggctcctggg caatatgatt gtgattgtgt tgggccacca cctgggcaaggaattcaccc 420 cctgtgcaca ggctgccttc cagaaggtgg tggctggagt ggccagtgccctggctcaca 480 agtaccacta aacctctttt cctgctcttg tctttgtgca atggtcaattgttcccaaga 540 gagcatctgt cagttgttgt caaaatgaca aagacctttg aaaatctgtcctactaataa 600 aaggcattta ctttcactgc 620 10 6 PRT Unknown OrganismDescription of Unknown Organism Hexapeptide 10 Val Tyr Ile His Pro Phe 15

1. A method of modulating neuronal activity, comprising the step of administering an effective amount of a neuroactive peptide having at least one of the biological activities of angiotensin IV as herein defined, comprising the amino acid sequence: Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, (SEQ ID NO:1), or a biologically-active analogue or fragment of said peptide, to a mammal in need of such treatment.
 2. A method of modulating neuronal activity, comprising the step of administering a biologically-active non-peptide analogue of the neuronal peptide according to claim 1 to a mammal in need of such treatment.
 3. A method according to claim 2, in which the biologically-active analogue is a peptidomimetic compound.
 4. A method according to any one of claims 1 to 3, in which the biological activity is selected from the group consisting of modifying learning, modifying behaviour, vasoactive effects, dilation of cerebral arteries, increase in renal blood flow, increase in stereotypy behaviour, facilitating memory retrieval, neurite modelling and alleviation of the effects of spinal cord injury.
 5. A method according to any one of claims 1 to 4, wherein said neuronal activity is selected from the group consisting of motor neuron activity, cholinergic neuron activity and neuronal development.
 6. A method of treating a condition selected from the group consisting of dementia; Alzheimer's disease; neuro-degenerative disorders involving one or more of cholinergic pathways, motor pathways, or sensory pathways; motor neuron disease; sensory peripheral neuropathies; motor peripheral neuropathies; brain injury; and spinal cord injury resulting from one or more trauma, hypoxia, and vascular disease, comprising the step of administering an effective amount of a neuroactive peptide having at least one of the biological activities of angiotensin IV as herein defined, comprising the amino acid sequence: Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, (SEQ ID NO:1), or a biologically-active analogue or fragment of said peptide, to a mammal in need of such treatment.
 7. A method according to claim 6, comprising the step of administering a biologically-active non-peptide analogue of the neuroactive peptide of claim 6 to a subject in need of such treatment.
 8. A method according to claim 7, in which the biologically-active analogue is a peptidomimetic compound.
 9. A method according to any one of claims 6 to 8, in which the biological activity is selected from the group consisting of modifying learning, modifying behaviour, vasoactive effects, dilation of cerebral arteries, increase in renal blood flow, increase in stereotypy behaviour, facilitating memory retrieval, neurite modelling and alleviation of the effects of spinal cord injury.
 10. A method according to any one of claims 1 to 9, in which the mammal is a human.
 11. A method of screening for putative agonists or antagonists of the effect of LVV-haemorphin-7 on neuronal activity, comprising the step of testing the ability of the compound to stimulate or inhibit the effect of LVV-haemorphin-7 on a biological activity selected from the group consisting of modifying learning, modifying behaviour, vasoactive effects, dilation of cerebral arteries, increase in renal blood flow, increase in stereotypy behaviour, facilitating memory retrieval, neurite modelling and alleviation of the effects of spinal cord injury.
 12. An antagonist of LVV-haemorphin-7, identified by the method of claim
 11. 13. An agonist of LVV-haemorphin-7, identified by the method of claim
 11. 14. A method of modulating neuronal activity, comprising the step of administering an effective amount of an antagonist according to claim 11 to a mammal in need of such treatment.
 15. A method of modulating neuronal activity, comprising the step of administering effective amount of an agonist according to claim 12 to a mammal in need of such treatment.
 16. A pharmaceutical composition comprising an agonist according to claim 11, together with a pharmaceutically acceptable carrier.
 17. A pharmaceutical composition comprising an antagonist according to claim 12, together with a pharmaceutically acceptable carrier. 